Control of Epstein-Barr virus infection by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) play a critical role in response to Epstein-Barr virus (EBV), a widespread human herpes virus that persists in healthy carriers as a latent infection of B cells and is implicated in the pathogenesis of lymphoid and epithelial cell malignancies. Six virus encoded nuclear antigens, EBNA1-6, and three membrane proteins, LMP1, -2A and -2B, are regularly detected in EBV transformed lymphoblastoid cell lines (LCLs). Recombinant vaccinia viruses expressing these genes were used to infect EBV negative cells that were then tested as targets for polyclonal EBV-specific CTL cultures. Five HLA A11 restricted epitope regions were mapped in the EBNA4 protein using overlapping synthetic peptides in blast sensitization assays. The cognate peptides of the immunodominant and subdominant epitopes were identified in residues 416-424 (IVTDFSVIK, designated IVT) and 399-408 (AVFDRKSDAK, designated AVF). The TCR alpha and beta chains of IVT- and AVF-specific clones derived from 4 donors were cloned and sequenced. The AVF specific responses was highly conserved with virtually the same TCR detected in all donors. In contrast, there was no preferential V(D)J usage and no obvious motif in the complementarity determining regions (CDR)3 loops of the IVT specific TCRs. Sequence variations was reflected in unique fine specificity patterns detected by Ala-scanning mutagenesis.

Analysis of TCR affinity as a function of sensitivity to CD8 blocking indicated that the diversity of the IVT specific repertoire does not reflect a compensatory expansion of low affinity clones due to elimination of a self-reactive high affinity TCR. Escape from CTL mediated rejection could play an important role for virus reactivation and spread and could also contribute to the pathogenesis of EBV associated malignancies. To address this question, the sequence of the IVT and AVF epitopes were analysed in virus strains isolated from populations with different HLA backgrounds. Isolates from 33 Southeast Asian individuals (>50% HLA A11 antigen frequency) had point mutations selectively affecting the codons for one of the IVT anchor residues in P2 or P9. In contrast only 4 of 15 isolates from Caucasians (10-15% HLA-A11 antigen frequency) and none of 15 isolates from Africans (HLA A11 practically absent) had mutations within the IVT sequence. Approximately half of the Southeast Asian isolates isolates carried mutations affecting anchor residues of the AVF epitope.